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ThesisItem Open Access ESTIMATION OF GENETIC DIVERSITY USING SSRs IN MAIZE INBRED LINES IN RELATION TO HETEROSIS AND COMBINING ABILITY(PAU Ludhiana, 2013) Nivgojind Singh Virk; S P S, BrarThe present investigation was conducted to estimate the magnitude of general and specific combining ability of inbred lines and crosses respectively and heterosis for yield and yield component characters of maize. Forty four F1 crosses were made in a line x tester fashion by using twenty two lines and two testers and all crosses with their parents were evaluated. The data was recorded on nine characters. The lines 3(LM13), 26-2009 and 37-2009 were good general combiners for most of the characters studied. The estimates of gca effects revealed that lines 10-2009, 22(LTP-4), 24-2009, 38-2009, 39-2009, 41(CML32), 95-2009 were good general combiners for grain yield/plant. The hybrids 29-2009xLM13, 37-2009xLM13, 97-2009xLM13, 9-2009xLM14, 97-2009xLM14 were found to be good specific combiners for most of the characters. Fourteen hybrids were found to be good specific combiners for grain yield/plant. The hybrids 3(LM11)xLM13, 3(LM11)xLM14, 13-2009xLM14, 22(LTP-4)xLM14, 24-2009xLM13, 24-2009xLM14, 37-2009xLM14, 39-2009xLM13, 41(CML32)xLM14, 45-2009xLM14, 46-2009xLM14, 59-2009xLM14, 95-2009xLM13, 95-2009xLM14 were found to be good cross combinations which showed significant mid parent heterosis for most of the characters. Unweighted pair group method with arithmetical averages (UPGMA) available in numerical taxonomic and multivariate analysis system (NTSYS-pc) 2.02e was used to group the inbred lines in different clusters. Dendrogram constructed grouped the inbred lines in two clusters (C-1, C-2). But the morphological grouping of parents was not in agreement with the heterotic pattern of their crosses. A set of twelve SSR primers dispersed throughout the maize genome was used to assess the genetic diversity at the molecular level. A total of forty alleles were detected and polymorphism information content (PIC) value ranged from 0.26 to 0.81 with an average value of 0.5. SSR primers with di-repeats revealed a maximum mean PIC value of 0.56, followed by tetrarepeats (0.44) and tri-repeats (0.39). It was able to assign inbred lines to three clusters (C-1, C-2 and C-3). In order to predict the hybrid performance, utilizing SSR markers, genetic distances were calculated among the inbred lines. Correlation coefficients calculated between the genetic distances of inbred lines and mid parent heterosis and specific combining ability for various yield and yield component characters of maize were non significant. Thus it seems rather difficult to make a generalized conclusion regarding the prediction of hybrid performance especially for grain yield, based on genetic distance measures.ThesisItem Open Access HETEROSIS, COMBINING ABILITY AND CORRELATION STUDIES IN THREE-WAY CROSS HYBRIDS IN SUNFLOWER (Helianthus annuus L.)(Punjab Agricultural University, Ludhiana, 2013) Yadvinder SinghStudy was undertaken to assess the magnitude of heterosis and combining ability in three-way cross hybrids of sunflower. A set of eleven A lines 302A X 11B, 302A X 42B, 302A X 234B, 302A X 44B, 44A X 40B, 11A X 234B, 43A X 44B, 234A X 40B, 44A X 234B, 44A X 42B, 44A X 11B and four restorers P89R, P69R, P93R and P110R were crossed in all possible combinations to obtain 44 TWC hybrids to evaluate for general combining ability (GCA) and specific combining ability (SCA) in a randomized block design over two years in spring 2011 and spring 2012. Genetic variability among genotypes was assessed for days to flower initiation, days to 50% flower initiation, days to maturity, plant height, head diameter, no. of leaves per plant, chlorophyll content, leaf area, leaf area index, seed yield per plant, 100 seed weight, oil content, stay green and fatty acid composition. CMS 11A X 234B had significant general combining ability effects for seed yield, head diameter, chlorophyll content, leaf area and oil content followed by CMS 44A X 40B for seed yield, plant height and days to flower initiation. Among the restorers P89R was good combiner for seed yield, head diameter, no. of leaves, chlorophyll content and 100 seed weight followed by P93R for head diameter, leaf area index, 100 seed weight and oil content. Two crosses 302A X 44B X P89R and 302A X 234B X P69R had significant sca effects for various characters. Importance of additive gene effects was found in the inheritance of traits like plant height and 100 seed weight. However for all other characters such as days to flower initiation, days to maturity, head diameter, no. of leaves, chlorophyll content, leaf area, leaf area index, seed yield and oil content non-additive gene effects predominated. Hybrids 302A X 44B X P89R, 11A X 234B X P89R and 234A X 302B X P93R excelled the standard checks for most of the important traits. The hybrids in general involving the parents 234A X 44B, 234A X 302B, P93R and P89R exhibited high positive heterosis for seed yield per plant. High significant correlation was observed between seed yield and 100 seed weight and plant height followed by head diameter, no. of leaves, chlorophyll content, leaf area and leaf area index. Keywords: Heterosis, combining ability and three way cross hybrid.ThesisItem Open Access GENOTYPE AND SITE INTERACTION STUDIES OF Salix CLONES IN PUNJAB(Punjab Agricultural University, Ludhiana, 2013) Singh, Kulwinder; Chauhan, RajniThe field trials were conducted during 2012-13 at three locations (environment 1: University Seed Farm, Ladhowal; environment 2: Badhian forest, Range and Division Dasuya and environment 3: Alamwala forest nursery, Range Malout, Division Faridkot) located in central plane zone, sub-mountain undulating zone and south-western zones of Punjab respectively. Twenty-two willow clones were planted in a randomized block design with three replications, to study the genotype and site interaction among different clones. The data for plant height and collar diameter was recorded at all the three locations and data for other parameters i.e., leaf parameters, leaf display, number of branches and insect/pest attack was recorded only at environment 1. All the characters under study at environment 1 exhibited significant differences among different clones. Differences among clones for plant height and collar diameter were significant under all three environments as well as after pooled analysis. On the basis of plant height and collar diameter, clone UHFS-13, UHFS-1 and UHFS-19 was best in environment 1, environment 2 and 3, respectively. Clone UHFS-1 and UHFS-19 showed wider adaptability. The values of genotypic variability for different traits were found between low to high and for phenotypic variability between moderate to high. Values of heritability and genetic gain were observed between low to high. On the basis of growth, number of branches leaf parameters and leaf display, clone UHFS-2, UHFS-5, UHFS-12 and UHFS-18 will be suitable for agroforestry, and clone UHFS-3, UHFS-13, UHFS-14 and UHFS-16 for biofuel or phytoremediation and Clone UHFS-10 for landscape purposes.ThesisItem Open Access Genetic analysis of hydrocyanic acid, water use Efficiency and quality traits over senvironment in forage sorghum [Sorghum bicolor (L.) Moench](Punjab Agricultural University, Ludhiana, 2013) goyalThe present investigation was undertaken in the Department of Plant Breeding & Genetics, Punjab Agricultural University, Ludhiana during 2010-2013 to study the genetic analysis of hydrocyanic acid (HCN), water use efficiency (WUE) and quality traits over environments viz. normal environment (Env-N) and water stress environment (Env-S) in forage sorghum [Sorghum bicolor (L.) Moench]. Under experiment-1, evaluation of 60 top crosses obtained from crossing 15 cms lines to four populations as males was done. Analysis of variance for green fodder yield and its component characters, HCN and traits affecting WUE revealed significant differences due to genotypes, environments and significant genotypic x environment interaction for most of the traits. The analysis of variance for combining ability suggested that the variance due to general combining ability (gca) and specific combining ability (sca) were significant and varied with the change in environment for important traits. The estimates of 2gca and 2sca indicated the predominance of non-additive gene action in the inheritance of all the traits. The cms lines, AKMS-14A, 2077A, NSS1007A and NSS1008A under Env-N and 940031A and NSS1008A under Env-S were good general combiners for green fodder yield and its component characters, low HCN content and high water use efficiency. With respect to green fodder yield, population RSSV-9 appeared to be the best general combiner under both the environments. One top cross 94002A x RSSV-9 had high sca effects for these traits in both the environments. Estimation of heterosis showed that three crosses viz. 94002A x RSSV-9 (54.15% and 27.25% under Env-N and Env-S, respectively), AKMS-14A x RSSV-9 (52.26% and 25.27% under Env-N and Env-S, respectively) and NSS1007A x Ramkel (48.49% and 7.69% under Env-N and Env-S, respectively) manifested significant positive heterosis over PSC-1 for green fodder yield and significant negative heterosis for HCN under both the environments. The selections for high green fodder yield under both the environments could be carried out by phenotypic selection for early vigour, plant height, leaf area index, photosynthetic capacity and proline content in the desired population. Under experiment-2, 100 families of each type viz. full sib, half sib and S1 were evaluated under two already mentioned environments. The trend for mean fodder yield and yield contributing traits viz. plant height, number of leaves per plant and leaf area index and HCN showed that the mean of full sib families exceeded the mean of half sib families and mean of S1 families. This indicated that the heterosis and inbreeding depression were important for these traits in this population. The green fodder yield and its components and traits affecting water use efficiency had 2A*/2A greater than 0.5 in both the environments. This suggested more contribution of additive gene effects than dominance effects for genetic control of green fodder yield and other important characters. For HCN content, the ratio of 2A*/ 2A was less than 0.5 in Env-N and greater than 0.5 in Env-S which suggested that dominance and/ or epistasis were more influential in controlling HCN content under Env-N. Additive genetic variation estimated appeared to be sufficiently great to permit substantial progress by any of the three family systems of selection, but S1 family selection appeared to be the most promising, particularly for improving green fodder yield, yield components, HCN content and WUE traits.ThesisItem Open Access MORPHO-AGRONOMIC AND MOLECULAR CHARACTERIZATION OF GENETIC DIVERSITY IN OAT (Avena sativa L.) GERMPLASM(Punjab Agricultural University, Ludhiana, 2013) Choudhary, Khushwant B.; Kapoor, RahulThe present study was undertaken to assess the genetic diversity present in the ninety six oat (Avena sativa L.) germplasm lines representing the collection from various eco-geographical regions of the country. On the basis of mean performance the lines viz; OL 1684 for plant height (121.80 cm), SKO 109 and OL 1693 for leaf length (69.65 cm) and leaf width (3.30 cm) respectively, OL 1615 and OL 1612 for GFY (89.50 and 88.00 kg/plot) and DMY (32.20 and 31.75 kg/plot) were found to be superior. Mahalanobis D2 analysis grouped all 96 genotypes into ten clusters. Cluster VII (26 genotypes) and Cluster IX (1 genotypes) were the largest and the smallest clusters respectively amongst all. The mean for GFY (84.50 kg/plot), DMY (29.84 kg/plot) was highest in cluster number V, whereas cluster VI had highest mean for leaf length (58.89 cm), leaf width (2.29 cm), days to 50% flowering (129.75) and days to maturity (159.75). The inter-cluster distance was highest between VI and IX cluster pair (25.87) followed by VI and X (21.51) suggesting significant high genetic diversity among genotypes of these clusters. The molecular diversity analysis using 40 SSR markers clustered all the 96 germplasm lines into ten clusters and significant level of dissimilarity (0.12 to 0.96) was depicted among the lines. Genotypic pairs having utmost genetic dissimilarity (0.96) were OL 1634 and OL1688, OL1702 and OL1688, OL 1705 and OL 1634, UPO 03-3 and OL 1688, UPO 03-3 and OL1705. PIC values ranged from as low as 0.06 to as high as 0.75 (AM 7). The primer pairs with highest PIC value were AM 7(0.75), AM2 (0.69) and AM10 (0.69). In addition, statistically non significant correlation with r=0.195 (P=0.05) was reported between morpho- agronomic and molecular diversity.ThesisItem Open Access STUDIES ON SOMATIC EMBRYOGENESIS AND GENETIC TRANSFORMATION IN MAIZE(Punjab Agricultural University, Ludhiana, 2012) Dhillon, Navjot KaurThe present investigation entitled, “Studies on somatic embryogenesis and genetic transformation in maize” was undertaken using maize inbreds viz., LM 5, LM 6, LM 13, LM 15 and LM 16. Callus induction was studied on ten media compositions based on MS (1962) salts using three explants, viz., immature embryos, mature embryos and split seeds. Among five maize inbreds thus investigated, LM 13 was most responsive to tissue culture. Highest (88.7%) callusing was observed from immature embryos cultured on MS + 2,4-D (3.0 mgL-1 ) + Picloram (10.0 mgL-1). Whereas, the maximum (88.6%) callus induction using mature embryos was observed on MS + Picloram (10.0 mgL-1) + BAP (0.5 mgL-1). Subcultured calli exhibited frequent somatic embryogenesis and the highest (72.6%) somatic embryogenesis was observed in inbred LM 13 on MS + Picloram (10.0 mgL-1) + BAP (0.3 mgL-1). The addition of proline, casein hydrolysate, silver nitrate and sucrose enhanced somatic embryogenesis in all the calli. Whereas, the cefotaxime did not have much effect on somatic embryogenesis. Embrogenic calli, upon their transfer to regeneration medium, exhibited shoot bud/shoot and root regeneration. The maximum (71.6%) shoot regeneration in LM 13 from immature embryo-derived calli was achieved on MS + Proline (3.0 mgL-1) + BAP (3.0 mgL-1) + Kin (0.5 mgL-1) + Gelrite (2.0%). Whereas, the mature embryo-derived calli of same inbred cultured on same regeneration medium exhibited poor (26.6%) shoot regeneration. Genetic transformation was attempted with particle gun (Bio Rad) using two target tissues viz. embroygenic calli and immature embryos. The bombardment was done using plasmids carrying glyI, glyII genes (for abiotic stress tolerance) and Cry1A(c) gene (for insect resistance). The bombarded tissues were selected using hygromycin (35 ppm) for two cycles of selection of two weeks each. In case of glyII gene bombardment, a total 146 plants were regenerated from which 42 plants (43.29%) were GUS positive. Further, the PCR analysis revealed the presence of glyII gene in 6 plants (4.10%). Whereas, in case of Cry1A(c) gene, a total 171 immature embryos were bombarded from which 2 plants (1.16%) were found to be PCR positive. The tissue cultured regenerated plants from all the bombarded tissues were grown in the transgenic glass house for collection of seeds. None of the plants carrying glyII gene set any seed. Whereas, four seeds have been collected from a plant carrying Cry1A(c) gene.ThesisItem Open Access Genetic Appraisal of Derived Brassica juncea Amphiploids(Punjab Agricultural University, Ludhiana, 2012) Gupta, MehakABSTRACT Derived Brassica juncea (AABB) lineages were analyzed to study the genetic changes that follow allopolyploid formations. Most of genotypes showed normal fertility with normal 18II chromosome configuration during meiosis1. Disomic chromosome substitution was indicated for DJ 15, DJ 18, DJ 19-2, DJ 21, DJ 38, DJ 58, DJ 61, DJ 92, DJ 97, DJ 103 and DJ 113. These substitution lines were expected to involve A/ C substitutions. Some individual plants showed novel phenotypes like determinate growth habit, terminal siliquae; however, there appeared to be no direct correlation between the examined molecular alterations and deviant phenotypes. There was a significant increase in sequence loss across generations within both subgenomes. There was a bias towards losing more sequences from the B. rapa (A) subgenome. We did not find any evidence of total sequence loss on the phenotypic means. Diversity analysis could also nicely resolve the test progenies into three very distinct groups, determinate juncea, crop Brassica parents and dipoid donors and indeterminate type of derived B. juncea. A total of 134 markers could be resolved on 18 linkage groups with an LOD score value greater than 3.0. The total genetic length of the map was 1010 cM with an average distance of 7.53 cM between adjacent loci. Resynthesis of new polyploid plants by derived resynthesis rout led to immediate and striking reactions of genomes to evolution. Gene loss, gene gain, gene movement and chromosome structural changes such as translocation and inversion were indicated and these collectively contributed to the process of diploidisation. A large number of hybrids were developed by hand crossing the set of derived B. juncea genotypes with natural B. juncea testers namely PBR210, RLC1, CBJ002 and AMH2. Heterosis over best commercial check PBR210, during 2010-2011 ranged from -76.5 to 103 percent with an average of -8.66 percent for the hybrids involving PBR 210 as male parent. The corresponding ranges for RLC1, CBJ002 and AMH2 were: -68.4 to 82.7 percent,-11.09 (60 hybrids), -83.6 to 79.6 percent, -3.96 (70 hybrids) and -78.3 to 59.0 percent,-14.53 (64 hybrids). During 2011-12, corresponding range and means for hybrids for involving same male parents were: PBR210 (-42.8 to 102.7; 16.87 for 35 hybrids); RLC1 (-52.7 to 104.8; 14.97 for 34 hybrids); CBJ002 (-55.9 to 117; 23.73 for 35 hybrids) and AMH2 (-51.8 to 69.6; 8.23 for 34 hybrids). Many of the putatic heterotic combinations will find their use in developing commercial hybrids. The concept of derived juncea developed by Brassica group at PAU stood validated by these studies.ThesisItem Open Access ALLELIC VARIATION, HETEROSIS AND COMBINING ABILITY IN OILSEED RAPE (Brassica napus L.)(Punjab Agricultural University, Ludhiana, 2013) Pratibha ChauhanBrassica napus (L.) accessions from China, Australia and India were characterized for diversity at morphological and molecular level, for assessing their use in crop improvement programs. ZY-014, BLN-3189, and BLN-3324 were identified to be high yielding genotypes. Analysis of variance confirmed the presence of morphological diversity for all the traits. D2 analysis allowed formation of nine broad groups, largely reflecting geographic origin. Allelic information from 36 polymorphic SSR primer combinations amplified 407 alleles and allowed clustering of genotypes into four distinct clusters. Australian genotype RT-108 was the most distinct genotype. Line x tester (38 x 4) was designed to estimate the potential of evaluated genotypes for hybrid breeding, and to identify promising parents combinations. Importance of non additive variation was emphasised. BLN-3348 was best combiner for yield followed by ZY-007, BLN-3224 and BLN-3575. Among testers, Rivette and GSL-1 were positive contributors for yield. Maximum positive SCA was recorded for CC-05032 X GSL-1 followed by ZY-014 X Rivette. Significant positive yield heterosis over commercial check was indicated in 29 combinations.BLN-3224 X Rivette showed the maximum commercial heterosis (80.52%). Yield heterosis was mostly associated with early flowering, late maturity, SPAD value, and leaf area and branch number. Genetic distance was significantly correlated with per se values for main shoot length, siliqua length and seeds per siliqua. Combined genetic distance based on both molecular and phenotypic diversity was correlated with heterosis for oil content. The diverse genotypes identified during the study represent a useful resource for improving heterotic potential and diversifying the B. napus gene pool at PAU, Ludhiana.ThesisItem Open Access EVALUATION OF CULTIVATED AND WILD ORYZA SPECIES FOR IRON UPTAKE UNDER AEROBIC CONDITIONS AND VARIATION IN IRON TRANSPORTER GENES(Punjab Agricultural University, Ludhiana, 2012) Rahul KumarThe cultivation of transplanted rice in northwest plains of India has lead to depletion of ground water to almost alarming level. Replacement of rice with other water requiring crop or changed production technology that will require less water has become essential. Cultivation of direct seeded rice under aerobic conditions is an alternative production technology that is expected to use less water, but it will require an altogether a different ideotype, one of the stresses the aerobic rice face is iron deficiency induced chlorosis. The present study focused on evaluation of cultivated and wild Oryza species for iron uptake under aerobic conditions and variation in iron transporter gene. Three hundred twenty Oryza accessions including six Oryza glaberrima, 105 O. rufipogon, 193 O. nivara, one barthii and 15 cultivated Oryza sativa genotypes were evaluated for iron deficiency induced chlorosis (IDC), days to 50% flowering; leaf area index and SPAD value. Some genotypes were tolerant to IDC throughout the growth period while other showed varying degree of chlorosis at the different stages. Based on the IDC score, 59 accessions including 49 tolerant and 10 susceptible were short listed for molecular studies and further evaluation. Basmati 370, Basmati 386, Lemont, PAU 201in cultivated varieties and O. nivara (CR 100003) and O. rufipogon (CR 100343) in wild species performed best under aerobic conditions with minimum or no chlorosis and desirable SPAD reading and LAI. These could be useful in aerobic rice breeding programme. In a hydroponics experiment O. rufipogon acc. CR 100001, O. nivara acc. CR 100127 and Basmati 370 showed better tolerance to IDC along with good SPAD value and high iron content in leaves. Microsatellite markers developed from 23 iron homoeostasis gene sequence were used for characterizing the 59 selected genotypes. Based on the variation in the 23 Fe homoeostasis genic SSRs markers, the 59 genotypes were clustered into three groups, significance of which is discussed.
